Factor Xa cleaves SARS-CoV-2 spike protein to block viral entry and infection

Serine proteases (SP), including furin, trypsin, and TMPRSS2 cleave the SARS-CoV-2 spike (S) protein, enabling the virus to enter cells. Here, we show that factor (F) Xa, an SP involved in blood coagulation, is upregulated in COVID-19 patients. In contrast to other SPs, FXa exerts antiviral activity. Mechanistically, FXa cleaves S protein, preventing its binding to ACE2, and thus blocking viral entry and infection. However, FXa is less effective against variants carrying the D614G mutation common in all pandemic variants. The anticoagulant rivaroxaban, a direct FXa inhibitor, inhibits FXa-mediated S protein cleavage and facilitates viral entry, whereas the indirect FXa inhibitor fondaparinux does not. In the lethal SARS-CoV-2 K18-hACE2 model, FXa prolongs survival yet its combination with rivaroxaban but not fondaparinux abrogates that protection. These results identify both a previously unknown function for FXa and an associated antiviral host defense mechanism against SARS-CoV-2 and suggest caution in considering direct FXa inhibitors for preventing or treating thrombotic complications in COVID-19 patients.

and 48 hpi (d) was determined by subsequently infecting Vero cells. All data are representative of three independent experiments. Data in c and d are presented as mean values ± SD and statistical analyses were performed by two-way ANOVA. PFU data were log2 transformed before running statistical models. Source data are provided as a Source Data file.

Supplementary Figure 3. FXa inhibits VSV-SARS-CoV-2 infection in MA104 cells while TMPRSS2, trypsin, and furin promote infection. (a and b)
Infectivity of VSV-SARS-CoV-2 in MA104 cells in the presence or absence of FXa, TMPRSS2, trypsin, or furin, determined by fluorescence microscopy. (c) VSV-SARS-CoV-2 was preincubated with furin, TMPRSS2, trypsin, or FXa at the indicated concentrations for 1 hour before it was used to infect M104 cells. Infectivity was quantified by flow cytometry at 48 hpi. (d) Virus titration by MA104 cells infected with VSV-SARS-CoV-2 in the presence or absence of FXa, TMPRSS2, trypsin, or furin was determined by subsequently infecting Vero cells. (e) VSV-SARS-CoV-2 was preincubated with MA104 cells for 1 hour, washed twice, and then exposed to treatment with FXa. Infectivity of the cells was quantified by flow cytometry at 24 hpi. All data are representative of three independent experiments. Data in b-e are presented as mean values ± SD and statistical analyses were performed two-sided Student's t test (e) or one-way ANOVA (b-d). MFI (b) and PFU (d) data were log2 transformed before running statistical models. Source data are provided as a Source Data file. Supernatants collected at 24 and 48 hpi were used to infect Vero cells for a virus titration assay. N=3 independent experiments. (c) VSV-SARS-CoV-2 was preincubated with plasma in which FX was converted or unconverted to FXa by incubating with FIXa and Factor V Activating Enzyme from Russell′s viper venom. Infectivity was measured at 24 hpi by fluorescence microscopy. N=3 independent experiments. (d) VSV-SARS-CoV-2 was preincubated with PBS, TF-FVIIa-FXa complex, FXa, FVIIa, or TF, followed by infecting M104 cells. Corresponding infectivity was measured by flow cytometry. N=3 independent experiments. Data are presented as mean values ± SD and statistical analyses were performed by two-sided Student's t test (c), one-way ANOVA (d), or two-way ANOVA (a, b). MFI (a) and PFU (bc) data were log2 transformed before running statistical models. Source data are provided as a Source Data file.

Supplementary Figure 5. FXa does not act on host cells but cleaves S protein expressed on cell surface, leading to shedding of S protein. (a)
MA104 cells were incubated with or without FXa for 1 hr, washed, and then infected with VSV-SARS-CoV-2 for 24 hr. Viral infectivity was measured by flow cytometry, measuring the expression level of GFP from virally infected cells. N=5 independent experiments. (b-c) The binding affinity of active FXa-Fc, inactive FXa-Fc, and Fc to full-length S protein (b) or VSV-SARS-CoV-2 chimeric viral particles (c) was quantified by ELISA. N=3 biologically independent samples. (d) The in silico prediction of the sites in the full-length S protein cleaved by furin, TMPRSS2, or FXa. Dark and light blue shades represent S1 and S2 subunits of the spike protein, respectively. (e-g) Shedding S protein by FXa. GFP and S protein surface expression on A549 cells, referred to as A549-S cells, treated with or without FXa were detected by flow cytometry (e). S protein from the supernatants of FXa-treated A549-S cells was measured by ELISA (f) and immunoblotting assay with an anti-S protein antibody (40591-T62, Sino Biological) (g). N=3 biologically independent samples. Data in g are representative of two independent experiments with similar data. Data are presented as mean values ± SD and statistical analyses were performed by one-way ANOVA. MFI data were log2 transformed before running statistical models. Source data are provided as a Source Data file. Figure 6. FXa cleavage blocks binding between S protein and ACE2. (a) Binding of S protein or FXa-pretreated S protein to the cell surface ACE2, determined by the amount of S protein on 293T cells expressing ACE2 using flow cytometry (left, a representative flow cytometry histogram; right, summary data). N=3 biologically independent samples. Data are presented as mean values ± SD and statistical analysis was performed by one-way ANOVA. MFI data were log2 transformed before running statistical models. (b) S protein was incubated with ACE2 for 1 hour, and then FXa was added to the incubation for another hour. Cleavage of S protein in the S protein-ACE2 complex by FXa was determined by immunoblotting with an anti-S protein antibody (40591-T62, Sino Biological). Data are representative of three independent experiments. Source data are provided as a Source Data file.

Supplementary Figure 7. The effect of direct and indirect FXa inhibitors on FXa-mediated inhibition of SARS-CoV-2. (a) Virus titration of FXa-pretreated vs. untreated VSV-SARS-CoV-2 in MA104 cells in the presence or absence of RIVA or FONDA was determined by re-infection of Vero cells with supernatants from MA104 infection. N=3 independent experiments. (b) S protein was incubated with
FXa that was pretreated with or without RIVA or FONDA. The binding capability of these treated S proteins with ACE2 expressed on 293T cells was assessed by flow cytometry (summary data of main Fig.  3l). N=3 independent experiments. Data are presented as mean values ± SD and statistical analyses were performed by one-way ANOVA (a, b). Source data are provided as a Source Data file. Cleavage of the D614G S protein by FXa was assayed by immunoblotting with an anti-S protein antibody (40591-T62, Sino Biological). (f and g) Binding affinity of ACE2 and the D614G S protein pretreated or not treated with FXa was measured by flow cytometry. N=3 independent experiments in (g). (h) Cleavage of WT WA1 S protein or Omicron S protein by FXa after 1-hour incubation was analyzed by immunoblotting using the same antibody in (e). All data are representative of at least three independent experiments. Data are presented as mean values ± SD and statistical analyses were performed two-sided Student's t test (c) and one-way ANOVA (a, b, g). Source data are provided as a Source Data file.
Supplementary figure 14. Flow cytometry gating strategy. Live cells were fist identified by low forward scatter (FSC) and low side scatter (SSC) gates. (a) To assay binding between the S protein and FXa, HEK293T cells expressing FXa were incubated with 10 µg/ml full-length S protein for 20 minutes at room temperature. Then the cells were washed twice and stained with anti-S protein antibody for 20 minutes at room temperature. After that, the cells were washed twice again and stained with an APClabeled secondary antibody (111-605-045, Jackson ImmunoResearch). Median fluorescence intensity (MFI) of APC was used to determine the binding capacity of FXa to S protein (Figs. 3b, 3j, Supplementary  Fig. 6a and Supplementary Fig. 13f). (b) To assay binding between the S protein-ACE2 complex and FXa, HEK293T cells stably expressing ACE2 protein were pre-incubated with the full-length S protein for 1 hr, washed twice with media, and then incubated with or without FXa for 20 minutes at room temperature. Cells were then washed twice and incubated with an anti-S protein antibody for 20 minutes at room temperature. Then the cells were stained with an FITC-labeled secondary antibody (111-005-003, Jackson ImmunoResearch) and washed twice, followed by flow cytometry assay. MFI of FITC was used to determine the binding capacity of FXa to the S protein-ACE2 complex ( Fig. 3d and Supplementary Fig.  13d).